ABSTRACT Disruption of intestinal epithelial barrier function is an important cause of HIV-associated chronic immune activation but underlying molecular mechanisms are not known. The central hypothesis of this proposal is that HIV infection impairs the regenerative capacity of intestinal stem cells, resulting in long-lasting barrier dysfunction. This hypothesis was formulated on the basis of available data in the literature showing that in inflammatory bowel diseases, the intestinal stem cell pool and the compositional balance of intestinal cell types is disrupted, and our own published results showing intestinal stem cell proliferation is impaired in a subgroup of HIV+ patients with high systemic inflammation and poor clinical outcome. Intestinal stem cell function can be tested in vitro by expanding them into self-organizing three-dimensional structures termed ?organoids?. They can be further differentiated into all intestinal epithelial cell types. This proposal leverages access to intestinal stem cells through intestinal biopsies by Dr. Ma Somsouk, an expert in HIV-associated gut inflammation, and expertise in organoid growth and basic HIV virology present in Dr. Melanie Ott's laboratory to examine intestinal stem cell function and organoid growth in HIV infection. The central hypothesis will be tested in two specific aims: 1) To define how HIV infection influences intestinal stem cell function in T cell: organoid co-culture experiments. The working hypothesis is that early contact between intestinal stem cells and HIV-infected T cells leads to stem cell damage, impaired gut epithelial regeneration and long-lasting barrier dysfunction. This hypothesis will be tested in T cell: organoid co-culture models established in the Ott lab. We will determine the effect of HIV-infected T cells on organoid growth, differentiation, barrier function and transcription at the single-cell level. Candidate factors such as TNF-? or factors emerging from our studies will be added directly to organoid culture media to analyze effects on growth and differentiation. 2) To determine the effects of chronic HIV infection on intestinal stem cell function. Our working hypothesis is that intestinal stem cell function is altered in HIV+ individuals, especially those with late onset of treatment and poor immunological recovery. This hypothesis will be tested by comparing growth and differentiation of organoids grown from HIV-infected individuals with different onset of treatment or uninfected individuals using morphological, functional and single-cell RNA sequencing analysis. This proposal will provide detailed insight into fundamental processes within the gastrointestinal tract that impact infection, persistence, and comorbidities in people living with HIV and is as such well aligned with this RFA. It is innovative because it shifts the focus to intestinal stem cells and their ability to renew the gut epithelium for proper barrier functions. It also uses innovative new organoid technology combined with clinical studies aimed at explaining why barrier defects persist in patients despite ART. Thus, important advances in the basic biology and novel therapeutic approaches towards HIV- associated chronic immune activation are expected.